Effect of a Ketogenic Diet on Alzheimer's Disease Biomarkers and Symptoms: ClinicalTrials.gov
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Effect of a Ketogenic Diet on Alzheimer’s Disease Biomarkers and
Symptoms:
“Brain Energy for Amyloid Transformation in AD (BEAT-AD)” Study
Protocol Identifying Number: ##
Principal Investigator: Suzanne Craft, PhD
Funded by: National Institute of Aging
Draft / Version Number: d.10.0
16 February 2018
Study Protocol – BEATAD Draft 16-February-2018 Page 1KEY ROLES Suzanne Craft, PhD, Principal Investigator Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-713-8832 suzcraft@wakehealth.edu Laura Baker, PhD, Co-Investigator Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-713-8831 ldbaker@wakehealth.edu Benjamin Williams, MD, Study Clinician Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-716-4101 benwilli@wakehealth.edu Iris Leng, PhD, Biostatistician Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-716-4564 ileng@wakehealth.edu Timothy Hughes, PhD, Co-Investigator Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-713-3851 tmhughes@wakehealth.edu Youngkyoo Jung, PhD, Co-Investigator Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-716-2837 yojung@wakehealth.edu Anthony Molina, PhD, Co-Investigator Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-713-8584 amolina@wakehealth.edu Chris Whitlow, MD PhD, Co-Investigator Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-716-5118 cwhitlow@wakehealth.edu Study Protocol – BEATAD Draft 16-February-2018 Page 2
Tina Brinkley, PhD, Co-Investigator Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-713-8534 tbrinkle@wakehealth.edu Jeff Williamson, MD, MHS, Co-Investigator Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-713-8565 jwilliam@wakehealth.edu Ashley Sanderlin, PhD, Co-Investigator Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-713-8817 asanderl@wakehealth.edu Kiran Sai, PhD, Co-Investigator Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-716-5630 ksolinga@wakehealth.edu Sam Lockhart, PhD, Co-Investigator Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-713-8145 snlockha@wakehealth.edu Siobhan Hoscheidt, PhD, Co-Investigator Wake Forest School of Medicine Medical Center Boulevard, Winston-Salem, NC 27157 P: 336-716-8702 shoschei@wakehealth.edu Study Protocol – BEATAD Draft 16-February-2018 Page 3
INTRODUCTION: BACKGROUND AND SCIENTIFIC RATIONALE
Overview
Dietary patterns have been linked to risk of developing Alzheimer’s disease (AD) – a
progressive fatal disease with no current effective treatments – that places an extraordinary burden on
patients, families and society. As recently reviewed, dietary patterns high in saturated fat and simple
carbohydrates, also termed “Western” diets, are associated with increased risk of AD and other
dementias, whereas diets high in mono- and poly-unsaturated fats, vegetables, fruits and lean proteins
are associated with reduced risk.1,2 It is not surprising that diet is a powerful modulator of brain aging.
The brain derives most energetic substrates such as glucose, amino acids and fatty acids directly from
dietary sources. Diet also impacts peripheral metabolism and may thereby affect the brain. For
example, the Western diet is associated with obesity, diabetes, inflammation, and vascular disease,
disorders that increase the risk of AD through multiple mechanisms. Diets have also been used as
therapeutic tools to treat medical conditions, such as the use of the ketogenic diet (KD) to treat
epilepsy, and the low fat diet to treat cardiovascular disease. In preclinical studies, elevating ketones in
AD mice improved memory and reduced levels of the toxic -amyloid peptide (A), a hallmark of AD
pathology.3-5. Studies that have elevated ketones with medium chain triglyceride (MCT) supplements in
humans with AD have also documented memory improvement.6,7
In the following section we describe research investigating the effects of the KD on the brain, and
support its investigation as a potential therapy for AD.
The Ketogenic Diet is a Powerful Modulator of Brain Function
The ketogenic diet (KD) is a very low carbohydrate, adequate protein diet developed by the Mayo
Clinic in the 1920’s to treat refractory epilepsy. Due to carbohydrate restriction, fats replace glucose as
a primary energy source; the liver converts fats into fatty acids and ketone bodies (-hydroxybutryate
and acetoacetate) that are readily transported into the brain. The KD is remarkably effective; 50-70% of
patients have >50% seizure reduction and 30% have >90% seizure reduction. 8 In recent years, several
adaptations of the KD have been developed that improve compliance and reduce health risks
associated with prolonged high intake of saturated fats. One such adaptation, the modified
Mediterranean ketogenic diet (MMKD), has comparable efficacy to the original KD but allows slightly
higher carbohydrate consumption to permit increased intake of vegetables and fruits, and emphasizes
fats and proteins derived from healthy sources such as olive oil and fish.
Mechanisms underlying the effectiveness of the KD are not definitively known, but candidates
include reduction of neuronal hyper-excitability through glutamatergic inhibition due to increased
production of GABA, and KD enhancement of mitochondrial metabolism with corresponding activation
of ATP-sensitive K+ channels. These and other potential KD-related mechanisms are discussed below.
KD Effects on Neuronal Excitability. Neuronal hyper-excitability, a cardinal feature in epilepsy, has
also been observed in amnestic mild cognitive impairment (aMCI) and early AD, particularly in brain
regions prone to early amyloid deposition. Such hyper-excitability has been proposed as a cause or an
effect of amyloid deposition, and may represent a compensatory response to reduced local ATP
production.9 Ketones modulate glutamate metabolism, increasing availability of the inhibitory
neurotransmitter GABA and may thus restore inhibitory-excitatory balance within the central nervous
system (CNS).10 Supporting this possibility, cerebrospinal fluid (CSF) GABA levels increased following
a 4-month KD intervention in epileptic patients, and greater GABA increase was associated with better
seizure control.11
Study Protocol – BEATAD Draft 16-February-2018 Page 4KD Mitochondrial Effects. Epileptogenesis is associated with disrupted mitochondrial energy
metabolism and increased reactive oxygen species (ROS), pathologies also related to AD.12
Mitochondria are the primary producers of ATP, through carbohydrate oxidation in the TCA cycle, and
through -oxidation of fats. Mitochondria also play critical roles in regulation of calcium and ROS.
Rodent studies have demonstrated that mitochondrial dysfunction promotes AD pathology.13-17
Importantly, mitochondrial bioenergetic deficits precede amyloid plaque formation in mice, 18 and
therefore offer a potential target for early intervention. A bioenergetic shift theory of AD has evolved
suggesting that the transition from normal aging to AD is associated with increasing reliance on fatty
acid oxidation to supply brain energetic needs, in part due to mitochondrial dysfunction.19, 20
In in vitro and preclinical studies, the KD has potent mitochondrial effects, modulating the expression
of enzymes involved in mitochondrial metabolism, as well as increasing mitochondrial biogenesis in key
brain regions such as the hippocampus.21 ROS are produced in the course of normal mitochondrial
metabolism, and may function as signaling effectors; they are maintained at non-pathological levels by
intact antioxidant defenses. Pathological ROS levels are thought to be both a cause and effect of
impaired mitochondrial metabolism, with glutathione (GSH) as a key ROS regulator. Ketones decrease
ROS levels in in vitro and preclinical models, in part due to increased GSH biosynthesis (for a review
see 12). In preliminary data, we observed that adults with aMCI treated with a 6-week MMKD had
increased mitochondrial activity in monocytes that correlated with changes in CSF A42 and memory.
Other KD-Related Mechanisms.
Protein Pathways - HIF-1, Sirt1 and MTOR: Ketogenic diets have been shown to impact other
pathways related to metabolism and autophagy. Ketogenic diets increased levels of hypoxia-inducible
factor-1 (HIF-1) in rodents, possibly due to succinate generation during mitochondrial metabolism of
ketone bodies. Increased HIF-1 in turn increased cortical capillary density through enhanced
transcription of vascular endothelial growth factor (VEGF), with associated memory improvement.22 The
KD upregulation of proteins Sirt1 and MTOR have also been reported to suppress A production and
tau hyperphosphorylation, with increased neuroprotection and autophagy.23, 24
KD Epigenetic Effects: A growing body of work documents KD effects on epigenetic
mechanisms. Increased neuronal hyper-excitability in rodent epilepsy models is associated with DNA
hypermethylation, which is reversed by KD.25 Such effects are mediated in part through -
hydroxybutyrate which functions as a histone deacetylase inhibitor,26 and through adenosine-mediated
normalization of DNA methylation.27 In our proposed study, we will examine KD epigenomic effects on
pathways related to oxidative phosphorylation, which in preliminary work we have shown to be related
to aging and cognitive function.
KD and CNS Insulin Resistance: Markers of brain insulin resistance have been documented in
neuropathological and ex-vivo stimulation studies of human AD brain, as well as in AD animal
models.28-30 Insulin plays an important role in memory as well as in regulation of -amyloid and tau
phosphorylation (for a review, see 31). The KD has dramatic effects on peripheral insulin sensitivity, and
in some rodent models also enhances brain insulin response and expression of brain insulin and IGF1
receptor mRNA,32, 33 although no changes were observed in a short-term diet in C57B6 mice34. New
methods using neural-derived plasma exosomes have been used to assess CNS insulin resistance;
exosome abnormalities indicative of CNS insulin signaling defects in adults with AD have been
reported.35
KD and Sphingolipid Metabolism: Recent metabolomic /lipodomic studies have identified
abnormal brain glycerophospholipids, sphingolipids, fatty acids, and their metabolites in AD and
aMCI.36, 37 Sphingolipids regulate cell membrane structure, function, and apoptosis. The coordinated
homeostasis between cholesterol and sphingolipids protects neurons, but also likely underlies some
Study Protocol – BEATAD Draft 16-February-2018 Page 5neurodegenerative processes in AD, as they both modulate amyloid precursor protein (APP)
processing.38-40 Ceramide is a key intermediate for sphingolipid metabolism.41 Elevated serum ceramide
and sphingomyelin predict incident cognitive impairment, dementia and hippocampal volume loss in
aging and aMCI.42-44 CSF ceramide and sphingomyelin also predicted CSF Aβ and tau levels in middle-
aged adults, suggesting they may also be biomarkers of early AD pathology.45 The KD has been shown
to reduce levels of ceramides and other toxic lipid metabolites.46 In the proposed study, we will conduct
targeted lipodomic and metabolomic analyses of sphingolipid, fatty acids and other pathways, in
addition to proteomic analyses, that may reveal novel biomarkers of early disease or treatment
response.
KD Effects in AD.
Preclinical Models and Effects on -Amyloid: Preclinical studies provide evidence of positive
effects of elevating ketones on AD pathological processes, and in particular on regulation of -amyloid,
which has been proposed as an initiator of the AD pathological cascade. Transgenic mice, 3xTgAD,
treated with a ketone-inducing intervention showed less amyloid and/or tau pathology, and improved
memory performance than chow-fed mice in three studies.3-5 These results are supported by findings
that ketones interact with A metabolism in multiple ways. A may be transported through axons into
intracellular compartments, where it interferes with mitochondrial function by binding to mitochondrial
proteins, leading to increase ROS. Ketones have been shown to reduce A neurotoxicity through
blocking A entry into neurons and reducing amyloid aggregation, with associated improvements in
mitochondrial function and memory.5 Furthermore, a KD has been shown to modulate gene expression
of - and -secretases, enzymes involved in Aβ clearance and degradation, and of genes involved in
mitochondrial biogenesis.19, 47 Although there is growing evidence of KD-A interactions, some studies
in mice have failed to demonstrate an impact, perhaps due to genetically-based differential
susceptibility to KD effects, as different mouse strains have been shown to have different metabolic and
seizure modulating effects of the KD.48,49
The preponderance of findings from preclinical studies demonstrates that ketones impact A
regulation, with associated effects on mitochondrial metabolism, ROS, and memory. Clearly, further
study is needed to demonstrate the relevance of these findings to humans. We have documented
changes in CSF A42 in adults with aMCI treated with a MMKD. In our proposed study, we will directly
address the question of whether MMKD modulates CSF A42 (our primary outcome) in humans.
Human AD Studies. Several studies have examined the effects of elevating ketones with
medium chain triglyceride (MCT) supplements in AD. An early study from our laboratory showed acute
memory improvement after MCT consumption in adults with early AD, an effect restricted to participants
without the APOE-e4 allele.7 In a Phase II double-blind, placebo-controlled, 3-month trial of a MCT
supplement in 152 adults with early AD, participants without the APOE-e4 allele showed significant
improvement on the Alzheimer’s Disease Assessment Scale-Cognition (ADAS-Cog). 6 This trial was
compromised however by a high rate of gastrointestinal side effects that necessitated a protocol
revision mid-trial, and resulted in a high drop-out rate in the active treatment group.
Use of a supplement to increase ketone availability is an attractive strategy due to its relative ease
of implementation. However, because supplements are consumed in the context of a normal diet their
efficacy may be attenuated. For example, MCT supplements do not reduce seizures in epilepsy.
Pathological aspects of the typical diet such as high simple carbohydrate intake may counteract KD
effects. Additionally, many nutrient-derived substances enter the brain through competitive diffusion,
and thus intake of competitive substrates may attenuate the benefit of ketone supplements. In contrast,
the KD promotes endogenous elevations of ketones that are readily transported into the brain. To date,
only one small trial has examined the effects of a KD in adults with aMCI.50 Twenty-three adults with
aMCI were randomly assigned to a 6-week KD or AHAD. The KD diet group showed enhanced verbal
memory, as well as improved peripheral metabolism.
Study Protocol – BEATAD Draft 16-February-2018 Page 6Summary: Strengths and Weaknesses of Supporting Research. The convergence of research to
date indicates that the MMKD, a KD variant designed to improve compliance and nutrition, is a potent
therapy for epilepsy and may benefit aMCI via multiple pathways, through: 1) reduction of neuronal
hyperexcitability; 2) improved mitochondrial function and cerebral bioenergetics; 3) reductions in
oxidative stress, inflammation and lipotoxicity; 4) enhanced autophagy; and ultimately, 5) improved A
and tau regulation. Preclinical studies in AD mouse models and preliminary studies that elevate
ketones in humans with supplements or brief diet intervention provide promising results. We will build
on this solid foundation to conduct a Phase II study that will provide rich data regarding the efficacy,
feasibility, safety, and underlying mechanisms associated with MMKD intervention. These data will
inform the design of a future Phase III trial and identify novel biomarkers and therapeutic targets that
may enhance precision medicine approaches to diet and AD risk.
OBJECTIVES AND PURPOSE
The purpose of this study is to examine the effects of a 4-month MMKD compared with an
American Heart Association Diet (AHAD - a regimen that has been shown to reduce the risk for
cardiovascular disease). We will investigate diet effects on AD biomarkers, on cognition, on
neuroimaging measures of metabolism, vascular function, and on CSF/blood epigenetic, exosome, and
omic markers. Our study will extend previous findings in several important ways by: 1) using a MMKD
rather than a traditional KD, which has the potential for greater long-term compliance and health
benefits; 2) increasing the sample size and duration of the diet intervention; 3) examining potential
mechanisms of diet effects that may result in new biomarkers and therapeutic targets; and 4) examining
key treatment response variables such as APOE genotype, amyloid positivity and metabolic status that
could inform precision medicine approaches to dietary prescription.
Objectives:
1. Assess the safety and feasibility of a 4-month MMKD vs. the AHAD in adults with aMCI, and
determine its effects on a key AD biomarker (CSF A42 – primary outcome), as well as on:
cognition, cerebral metabolic rate of glucose utilization (CMRg) measured with 18F-FDG PET;
brain ketone utilization assessed with 11C-acetoacetate PET; MRI measures of perfusion,
hippocampal volume, default mode connectivity, and white matter integrity; and other CSF
biomarkers (total tau, p-tau181).
2. Investigate response moderators: APOE genotype, amyloid PET positivity and metabolic status.
3. Investigate proposed mechanisms underlying MMKD effects, including: 1) neuronal activity and
synaptic function (MRI, PET); 2) bioenergetic pathways (mitochondrial function assays, plasma
neural-derived exosome markers); 3) epigenetic analyses; 4) CSF/blood multi-omic panels
(proteomics, metabolomics, lipidomics, transcriptomics).
Importance of the Knowledge to be Gained
In addition to knowledge gained about the potential therapeutic efficacy of a Modified
Mediterranean-Ketogenic Diet intervention in adults with early AD, the trial will also provide important
knowledge concerning the role of glucose and lipid metabolism in AD and in the effectiveness of diet-
induced ketosis. Baseline metabolic status and changes in metabolic profiles may provide insight into
the differences between baseline status of the groups, but also response to the proposed intervention.
This metabolic profile analysis may ultimately be used as a biomarker to determine potential efficacy of
similar interventions in AD. Such knowledge will be of great utility in the design of other therapeutic
interventions. Based on these potential contributions, and the minimal potential risks to safety of the
participants, the risks of the trial are reasonable.
Study Protocol – BEATAD Draft 16-February-2018 Page 7STUDY DESIGN AND ENDPOINTS
Adults with aMCI (n=120) will be randomized on a 1:1 schedule to receive either a 4-month
MMKD or AHAD intervention. The MMKD and AHAD macronutrient profiles will be identical to those
tested in a pilot study (IRB# 0029992). Diet interventions will be equicaloric with participants’ normal
diets. Personalized nutritional guidance and menus will be provided, and compliance will be assessed
by a registered dietitian. Amyloid PET will be conducted at baseline. Blood collection and cognitive
assessment will be conducted at baseline and after 2 and 4 months of diet. Lumbar puncture (LP), MRI
and dual tracer PET will be conducted at baseline and following the intervention for all participants (see
Table 1. for a list of study measures).
Participants
Participants will be recruited from the Wake Forest Alzheimer’s Disease Core Center (ADCC), and from
the community. Participants aged ≥55-85 years will be enrolled who meet adapted NIA-AA criteria for
aMCI 51, and adjudicated by expert multidisciplinary consensus. The criteria for aMCI will be based on
cognitive and clinical evaluation, and all cognitive and clinical data will be considered in assigning
diagnoses. In general, cognitive testing will indicate two deficits, 1 SD below age and education-
adjusted means, on two memory tests of free and delayed recall, or on one memory and one language
or executive function test. In addition to numeric criteria, expert clinician judgment will be used to
interpret cognitive performance. The CDR score for study inclusion may be 0 or 0.5, and subjective
cognitive complaints will not be required from the participant or study partner. Given that we will
conduct amyloid PET, we will be able to determine which participants meet criteria for aMCI due to AD.
51
We will not restrict enrollment on this basis, but rather use this criterion in responder analyses.
Sample Size
120 participants will be recruited for this study. Males and females will be comparably represented, and
every effort will be made to enroll an ethnically diverse sample that typifies the geographical region.
Participants will be randomized to one of two diets (MMKD or AHAD) for four months, with 60
participants in each diet group.
Length of Intervention
We have observed effects on CSF biomarkers, cognition and imaging in our pilot study with a 6-week
MMKD intervention, and in previous month-long diet interventions. 52 We believe that a 4-month period
will provide important feasibility and compliance data, as well as potentially reveal other cognitive and
biomarker effects useful for the design of a Phase III study.
AHAD as a Control Diet
We believe that the AHAD is a good comparator for the MMKD because it will require the same
attention to food intake and thus control for “meta” aspects of the intervention. As a healthy diet it is
reasonable to expect it may benefit some participants, and thus will equate expectations across the two
diet intervention groups. In our pilot study the AHAD diet produced a distinct profile from the MMKD,
and thus will allow us to investigate response predictors that may inform precision medicine dietary
prescriptions.
Study Endpoints
CSF A42 as a Primary Endpoint: The convergence of findings from preclinical studies demonstrate
that elevating ketones impacts A regulation, and we have documented changes in CSF A42 in aMCI
treated with MMKD in our pilot study. Using CSF A42 as a primary outcome will allow us to address
the important question of whether the MMKD modulates AD pathological processes. Given that aMCI is
Study Protocol – BEATAD Draft 16-February-2018 Page 8reliably associated with reduced CSF A42, the expected direction of a beneficial response is for CSF
levels to increase, as we have observed. We will be able to support the interpretation that increased
CSF A42 reflects benefit by examining associations with other outcomes. We have extensive
experience in recruitment for studies requiring LPs; we successfully recruited 100 participants for a
recent trial of a Western diet that required 2 LPs (R37AG-10880), and our pilot trial required 3 LPs. The
fact that 97% of our participants return for follow-up LPs attests to our excellent safety record and the
skill of our clinicians in minimizing discomfort.
Preclinical Alzheimer Cognitive Composite (PACC) as a Cognitive Endpoint: Although the PACC was
designed for use with preclinical AD, it is reasonable to assume it will also be sensitive in aMCI, as all
PACC components are frequently used in aMCI studies. According to PACC developer, Reisa Sperling,
MD, it is a useful indicator of cognitive change in aMCI in the Harvard Aging Brain Study (personal
communication). Further, we observed significant improvement in our cognitive test composite that
includes 2 PACC components following the MMKD.
Selection of Endpoints
We have designed an ambitious study that will collect data on multiple outcomes, to inform future trial
design and to enable the identification of novel biomarkers and therapeutic targets. The approach we
have taken is to identify a limited number of analytics from each of the proposed methods that are well-
supported by our pilot data and that will be analyzed as part of the project (Table 1. below).
Table 1. All Study Endpoints
Primary
CSF AB42
Secondary
CSF Total tau, p-tau 181, ceramides, fatty
acids, redox proteomic profile
PET Global 11C-AcAc uptake, 18F-FDG
ALZ/PMOD
Hippocampal volume, AD signature
cortical thickness, Default Mode
MRI
Network connectivity, PCASL
posterior cingulate perfusion
Mitochondrial basal, maximal, spare
respiratory capacity, OXPHOS
Blood transcriptomic index, OXPHOS
methylation index, exosome insulin
resistance index
PACC (Primary), ADAS-COG 12, CDR,
Cognition/Function Executive function tests, Cogstate
tests, ADCS-ADL
NPI-Q, Sleep Quality Assessment
Mood/Sleep (PSQI), Epworth Sleepiness Scale,
WatchPAT
Setting: All study visits will take place at Wake Forest Baptist Medical Center.
Study Protocol – BEATAD Draft 16-February-2018 Page 9STUDY ENROLLMENT AND WITHDRAWAL
Participant Inclusion Criteria:
1. Male or post-menopausal female;
2. Age 55 to 85 years inclusive;
3. Diagnosis of amnestic mild cognitive impairment (aMCI; single or multi-domain);
4. An informant (study partner) able to accompany the participant to at least 3 visits, including at
least two diet education visits;
5. Stable medical condition (generally 3 months prior to screening visit) at the discretion of study
physician;
6. Stable on medications (generally 4 weeks prior to screening visit) at the discretion of study
physician;
7. Able to complete baseline assessments;
8. Fasting glucose within the normal range.
Participant Exclusion Criteria:
1. Diagnosis of neurodegenerative illness (except for MCI);
2. History of a clinically significant stroke;
3. Current evidence or history in past year of focal brain lesion, head injury with loss of
consciousness or DSM-IV criteria for any major psychiatric disorder including psychosis, major
depression, bipolar disorder, alcohol or substance abuse;
4. Sensory impairment (i.e.: visual or auditory) that would preclude the participant from
participating in the protocol;
5. Diabetes that requires current use of diabetes medications;
6. Clinically significant elevations in liver function tests;
7. Active neoplastic disease (stable prostate cancer and non-melanoma skin cancer is
permissible);
8. History of epilepsy or seizure within past year;
9. Contraindications for MRI (claustrophobia, craniofacial metal implants, pacemakers);
10. Significant medical illness or organ failure, such as uncontrolled hypertension or cardiovascular
disease, chronic obstructive pulmonary disease, liver disease, or kidney disease;
Study Protocol – BEATAD Draft 16-February-2018 Page 1011. Use of the following medications: anticonvulsants, drugs with potential interfering CNS effects
(other than cholinesterase inhibitors or memantine), medications with significant anticholinergic
activity, anti-parkinsonian medications or regular use of narcotic analgesics; small stable doses
may be permitted at the discretion of study clinician
12. If female, menstruation in the past 12 months or hysterectomy and current hormone
replacement therapy medication;
13. Major digestive disorders, absorption issues, or surgeries that may be exacerbated by diet
changes;
14. Untreated hypothyroidism or B12 deficiency;
15. Participants currently using resveratrol, CoQ10 (coenzyme Q10), coconut oil/other medium
chain triglyceride-containing (ie: Axona) supplements, or curcumin will be excluded unless they
are willing to discontinue them 2 weeks prior to the start of baseline visits and remain off for
study duration.
Strategies for Recruitment and Retention
A total of 120 participants (age 55-85 years) will be recruited for completion of the proposed study.
Subjects that meet inclusion criteria will be randomized into one of two diet groups, the MMKD or
AHAD. The AHAD group will serve as the control group and the MMKD the intervention group, each
with a target of 60 participants.
A standardized phone screen questionnaire will be used to determine basic eligibility. Potential
participants who pass this initial screen will then receive an in-person screening, consisting of
physical/neurological exam, medical history, and neuropsychological exams to confirm an aMCI
diagnosis. All clinical data will be reviewed by expert study physicians and neuropsychologists. Clinical
laboratories and physical/neurological exam will be performed, or may be reviewed from a clinic visit
within the last 6 months, in order to exclude patients with major medical disorders.
Additionally, participants that gave blood in the past 56 days must agree to abstain from donating blood
for the duration of the study. Study visits will not be scheduled until 56 days after the most recent
donation.
Subject Recruitment Methods
Our lab has had ample success in recruitment for its clinical studies, thus current recruitment methods
for other lab studies will be used. These methods include newspaper advertisements in the Winston
Salem Journal and The Chronicle, radio advertisements, postcards, website, the Memory Assessment
Clinic (MAC) at Wake Forest Baptist Medical Center and community talks in churches and retirement
communities. Males and females will be comparably represented, and every effort will be made to
enroll an ethnically diverse sample that typifies the geographical region. Recruitment through talks and
print/radio ads will be targeted at the minority populations and women in order to ensure representative
enrollment in these populations.
Once participants have been identified as potential study candidates, they will be contacted by
telephone by the study coordinator. The study coordinator will explain the nature of the study and the
study procedures by reviewing the material presented on the consent form. Potential participants who
express interest in the study will be asked to meet with the coordinator and a study nurse, at which time
the consent form will be reviewed again and questions or concerns about the study will be addressed.
Study Protocol – BEATAD Draft 16-February-2018 Page 11Potential participants for this trial may also be recruited through other studies ongoing in our lab, including the Alzheimer’s Disease Core Center (ADCC). Participants who have participated in other clinical trials, such as the ADCC study, within the past 3 months will not have to repeat clinical labs, screening cognitive tests, MRI, LP, and DEXA during their screening/baseline visits. The data previously collected for the ADCC study will be used. DIET INTERVENTION Interventions and Interactions All potential participants for the study will first undergo a screening visit. Upon enrollment participants will have a total of three Core study assessments (data may be collected over several days for each Core assessment as long as visits are completed within two weeks – imaging, LP, etc.) occurring at baseline, 8 and 17 weeks (on-diet assessments), and 24 weeks (post-diet follow-up assessment). In addition to Core study visits participants will have weekly diet education visits for the first eight weeks, then return at 12 weeks to assess diet compliance over the course of the diet intervention. On week’s 9-11 and 13-16 participants will have weekly telephone contact with the study dietitian to discuss food selection, meal preparation and any issues with diet compliance. Experimental Diet We will use a Modified Mediterranean-Ketogenic Diet (MMKD), which is a very low carbohydrate diet (
weeks; food diaries will be completed at least 3x per week and include food/drink description and volume. Capillary ketone and glucose levels will also be measured in person weekly for the first two months, then monthly. If compliance is not satisfactory based on ketone measurements or food diary review, weekly in-person visits will be reinstated. Participants will be required to supply their own food based upon a daily meal plan, food list, and other educational material provided. A food stipend of $25 per week will be given to participants to help defray food cost, and a food scale to aid in food preparation. Participants will be asked to keep their exercise stable throughout the study. We have used these methods in our pilot study and found that they were highly successful in achieving dietary targets and maintaining compliance. Less than 10% (2 of 21) of participants discontinued our pilot study and the remaining participants showed good compliance. Because participants will be encouraged to maintain their normal caloric intake, they will experience minimal weight change throughout the intervention. We have defined weight stable as ±5 pounds. Weight will be monitored at each study visit, and caloric recommendations adjusted to ensure weight stability. Storage and Distribution of Study Supplements The multivitamins and olive oil provided to participants in the study diet groups will be stored within the Sticht Center at Wake Forest University School of Medicine. The supplements will be kept within a locked area on the first floor Kulynych Research Center. Participants on the MMKD will receive olive oil as needed at their in-person study visits. The olive oil will be used as an added fat source. All participants in the diet study will receive Centrum Silver multivitamin tablets at baseline to take throughout the study. Blinding: Although it is not possible for participants and the dietitian to be blinded to the study diet, all study personnel involved in performing cognitive testing or other outcome procedures or data analysis will be blinded. Participants will receive equal attention and instruction regarding their diets, and must expend similar amounts of effort to comply with dietary requirements. It will also be emphasized to participants that both diets may reasonably be expected to provide health benefits. STUDY PROCEDURES AND SCHEDULE Screening and Enrollment: Following a 12-hour fast, prospective participants will arrive at the Clinical Research Unit at the Sticht Center for Healthy Aging and Alzheimer’s Prevention and sign informed consent. Participants will then undergo the following screening procedures to confirm eligibility: brief cognitive screening assessment, blood draw for screening labs (CBC, CMP, LFTs, lipid panel, calcium, thyroid stimulating hormone (TSH), B12, and PT/INR), blood pressure, height/weight, and a brief physical exam and questionnaires. Following the screening visit, the eligibility of the participant for enrollment will be determined by the study physician and an expert consensus panel. Once a participants has been enrolled they will be asked to complete a physical activity log and 3-day food diary prior to their first diet education visit. These measures will be used to determine basic information about physical activity level, chronic caloric intake and typical pre-study dietary habits. Participants will continue to adhere to a self-selected, ad-libitum diet until the diet intervention begins. Note: Screening data from other center studies may be used if a prospective participant is interested in the study. These data are valid as long as it was acquired within 6 months of the baseline visits for this diet study. Core Study Visits: The Core study visits will be scheduled to begin within a two-week period before the diet intervention begins. The Core study visits include the Pre-Diet visits, Mid-Diet visit, the Post- Diet visits, and the Post-Diet Follow-up visit. The Core study visits will include a combination of the Study Protocol – BEATAD Draft 16-February-2018 Page 13
following study measurements: DEXA scan, amyloid and dual tracer PET, structural/functional MRI,
lumbar puncture (LP), cognitive testing, blood draw, capillary glucose/ketone testing, and stool
collection.
Pre-Diet Study Visits: The Pre-Diet Study Visits will occur during 2 weeks prior to the start of
the assigned study diet. At these visits, cognitive assessment, blood testing, capillary
glucose/ketone testing, MRI, PET, DEXA, stool collection, and LP will occur. If the participant
has not had the Apolipoprotein E (APOE) genetic test performed in a previous study, we will ask
that they agree to the test and draw a sample for that purpose at the Pre-Diet visit. Once
participants complete the Pre-Diet Lumbar Puncture (Visit 3) they will be randomized to a diet
group.
Mid-Diet Study Visit: The Mid-Diet visit occurs at week 8 (Visit 13) and will include diet
education and compliance check, cognitive testing, stool and blood sample collection. At this
visit additional measures will be recorded such as capillary glucose/ketone testing, weight and
metabolic outcomes.
Post-Diet Study Visits: The Post-Diet Study Visits will occur during week 17 (Visits 15-17). At
these visits, cognitive assessment, blood testing, capillary glucose/ketone testing, MRI, PET,
DEXA, stool collection, and LP will occur.
Post-Diet Follow-up Visit: The Post-Diet Follow-up Visit will occur during week 24 (Visit 19)
after participants have discontinued the diet and returned to an adlib diet. At this visit, cognitive
assessment, blood testing, and capillary glucose/ketone testing will occur.
Diet Educational Study Visits: Over the course of the diet study, participants whether on the MMKD
or AHA diet, will participate in a series of individual, group and phone educational visits to inform
participants of diet specifics, develop personalized meal plans, ensure adherence to their diet, and
answer any questions. The study dietician will lead these educational sessions in the clinic or by phone.
In-Person Diet Education Sessions: The Pre-Diet Education session will take place within 1
week prior to the start of the diet (90 min session). After diet initiation, for both diets, participants
will meet with the study dietitian weekly for the firest 8 weeks of the study for continued diet
education and assessment of compliance. Participants will return to the clinic at week 12 for diet
education and compliance check.
If participant enrollment and scheduling permit group sessions, participants may meet as a
group or individually during later weeks. These sessions will last from 30 min (individual) to 60
min (group) each and will provide participants support and the opportunity to ask questions. Diet
guidelines will be reviewed at each session. However, if necessary to improve diet adherence
and understanding, individual sessions with the study dietitian may take place as needed during
the remainder of the study. The need for these sessions can be determined by the study
dietitian and/or by participant request.
Phone Sessions: Each participant will have a phone session in addition to diet education with
the study dietitian 3-4 days after starting the diet and again in week 2. These session will allow
the participants and caregivers to ask questions and receive support as needed. Phone
sessions will also occur in weeks 9-11 and 13-16, and may be increased in frequency as
needed to optimize compliance.
Note: Data will be recorded at Diet Education Visits, including capillary glucose/ketone testing,
weight, adverse events, diet compliance, and nutritional intake.
Study Protocol – BEATAD Draft 16-February-2018 Page 14Table. 2. Schedule of Study Visits and Procedures
Clinic Visit # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Visit Week -12 Baseline 1 2 3 4 5 6 7 8 12 17 20 24
Pre amyPET
Post dtPET
Pre dtPET
Interm. F/U
Post Cog
F/U Visit
Mid Cog
Pre Cog
Post LP
Screen
Pre LP
Edu
Edu
Edu
Edu
Edu
Edu
Edu
Edu
Visit Name
Informed Consent X
Demographics X
Medical History X
Phys/Neuro Exam X
Screen Cognitive Battery X
Concomitant Meds X X X X
Resting Metabolic Rate X X
Vital Signs X X X X X X X X X X X
Diet Cognitive Battery X X X X
Weight X X X X X X X X X X X X X X X
Capillary Gluc/KB X X X X X X X X X X X X X X
Blood Draw: X X X X X X X X X
APOE Genotyping X
Clinical / Safety Labs X X X X
Metabolic Outcomes X X X X X X
Mito, Epign & Exo X X
Blood Banking X X X X X X
Stool Banking X X X X
CSF Banking X X
LP X X
DEXA Scan X X
MRI X X
dtPET X X
amyPET X
Diet Edu. / Compliance X X X X X X X X X X
Food Diary X X X X X X X X X X X
Compliance Call X X X X
Adverse Events X X X X X X X X X X X X X X X X X X
Safety Phone Check X X
Sleep Battery/Monitor. X X X X
Study Protocol – BEATAD Draft 16-February-2018 Page 15Study Visit Measures
Clinical/Physical Evaluation: During eligibility screening, participants will receive standardized clinical
cognitive evaluation (ADCC UDS3), a clinical interview and physical/neurological exam, clinical labs,
and depression screening with the Geriatric Depression Scale (GDS). 53
Metabolic Outcomes: Fasting plasma or serum will be sent out for assay by LabCorp diagnostic
company for glucose, insulin, and lipids (total cholesterol, LDL, VLDL, HDL, triglycerides) at screening,
baseline, month 2, and month 4. Quick analysis of metabolic outcomes done in week 1 and 12 or to
ensure participant safety may be delivered to the Wake Forest CLIA-certified laboratory for expedited
processing. Capillary ketone levels and weight will be measured at each in-person visit in the center.
Body composition will be measured using DXA at baseline and at the end of the diet intervention.
Cognitive Testing Visits: For all cognitive visits, participants will be asked to eat a standard meal the
night before and fast for 12 hours prior to start of visit. Upon arrival, weight and vital signs will be
obtained and then blood will be drawn (about 3 tablespoons). A breakfast meal will then be provided
(standard meal for baseline visit and study meal (MMKD or AHAD) for other visits) and the cognitive
protocol will begin. All cognitive test administration will be audio-recorded.
Cognitive Battery: The cognitive protocol will consist of both computerized and paper-based
assessments and will include all components of the memory composite as well as additional
assessments. Cognitive testing will be audiotaped using a digital recorder for quality assurance and
random checks will occur for quality control and to correct drift. Recordings will be erased once review
has been completed. The total cognitive battery will take approximately 60-75 minutes to administer at
both screening and Core Study Visits. Methods for cognitive assessments and other evaluations are
described in this section.
Screening Cognitive Battery:
Participants will undergo a cognitive assessment at screening using the Uniform Data Set
(UDS) version 3 test battery. Objective tests of cognitive function will include the following:
global cognitive function (MOCA, MMSE), CDR, premorbid cognitive function (AMNART, Rey
Auditory Verbal Learning Test), memory (Craft Story), visual memory (Benson Complex Figure),
attention/concentration (forward and backward Number Span, Trails A/B), and language/verbal
fluency (Verbal/Category Fluency, Multilingual Naming Test).
Diet Cognitive Battery
Once participants have been enrolled in the study they will complete another cognitive battery that will
measure the effect of diet on cognition. The Diet Cognitive Battery will consist of the Preclinical
Alzheimer Cognitive Composite (PACC), CogState computerized test battery, the Alzheimer’s Disease
Assessment Scale – cognitive subscale (ADAS-cog) version 12, the Alzheimer’s Disease Cooperative
Study – Activities of Daily Living Scale (ADCS-ADL), and the Geriatric Depression Scale (GDS). The
major components of each battery are outlined below:
1. PACC (primary cognitive endpoint):
i. Free and Cued Selective Reminding Test – Total Recall: The FCSRT
measures verbal memory through controlled learning of a list 16 words that are
introduced to participants by both visual and auditory presentations. Once the
participant has learned the list they immediately recall each item. Then after a
Study Protocol – BEATAD Draft 16-February-2018 Page 16delay of 20 minutes participants are asked to recall the items over three trials.
The total score is the sum of free and cued words recalled over all three trials.
ii. Logical Memory IIa – Delayed Paragraph Recall: Participants will be read a
story divided into ‘bits’ of information containing main content words. The
participant recalls the story immediately, then again after a 20 minute delay. The
total number of ‘bits’ remembered after the delay will be calculated as the
delayed recall score.
iii. Digit Symbol Substitution Test: The participant will be introduced to a sheet of
paper with a key that matches numbers 1-9 with an unfamiliar symbol. The
participant must work as fast as possible for 90 seconds to match a random list
of numbers 1-9 with their associated symbol.
iv. MMSE – Total Score: The MMSE is a questionnaire read to participants and
tests multiple areas of cognition. The total number of questions right indicate
general cognitive ability with higher numbers representing higher cognitive
function.
v. Category Fluency: Participants will be given a category then asked to verbally
generate nouns that belong to that category. The total number of correct items
generated in 60 seconds will be summed.
2. CogState Computerized Test Battery:
i. Tests of Learning and memory
i. Object Pattern Separation Task (OPST), target pictures of common objects are
presented along with highly similar “lures” and participants must identify targets;
ii. One Card Learning (OCL) Test, a playing card is presented and the participant
must decide whether he/she has been shown that card before. The arcsine
proportion correct is calculated
ii. Tests of psychomotor speed:
i. Detection Test (DT), as soon as a playing card is presented, the participant must
press the "Yes" key;
ii. In the Identification Test (IDT), the participant must decide whether the card
presented is red. Number correct and/or mean correct response time in log
(msec) are recorded.
3. ADAS-Cog 12: The ADAS-Cog 12 will also be administered to provide data to inform future
trial design. The ADAS-Cog 12 is a psychometric instrument that evaluates memory, attention,
reasoning, language, orientation, and praxis. A higher score indicates more impairment. Scores
from the original portion of the test range from 0 (best) to 70 (worse) and then number of items
not recalled ranging from 0-10 is added for a maximum score of 80. A positive change indicates
cognitive worsening. The ADAS-Cog12 version will be used in the current study, which includes
Delayed Word Recall - a measure of episodic memory.
4. ADCS-ADL: The Alzheimer’s Disease Cooperative Study - Activities of Daily Living Scale
(ADCS-ADL) is an activities of daily living questionnaire aimed at detecting functional decline in
people with Mild Cognitive Impairment (MCI). It was adapted from an inventory developed by
the ADCS to assess functional performance in participants with Alzheimer’s disease. In a
structured interview format, informants are queried as to whether participants attempted each
item in the inventory during the prior 4 weeks and their level of performance. The ADCS-ADL-
MCI scale discriminates well between normal controls and patients with mild AD or MCI. It has
good test-retest reliability. The questions focus predominantly on instrumental activities of daily
living scales (e.g. shopping, preparing meals, using household appliances, keeping
appointments, reading).
Study Protocol – BEATAD Draft 16-February-2018 Page 175. Geriatric Depression Scale: The Geriatric Depression Scale (GDS) is a well-validated self-
report questionnaire used frequently in clinical and research settings to assess mood in older
adults. This questionnaire includes 15 questions and takes about 5-7 minutes to complete. The
GDS will be used as a part of the screening cognitive battery to determine whether participants
should be excluded due to a significant presence of depressive symptoms.
6. Neuropsychiatric Inventory Questionnaire: The neuropsychiatric inventory questionnaire
(NPI-Q) is widely used to assess the presence of mood disorders in geriatric populations. The
NPI-Q measures 12 symptom categories and is scored on a scale of 0-36. An informant or
caregiver for the participant must answer each question by indicating Yes or No for the
presence of a symptom and when present they rate the severity on a scale of 1-3.
Imaging Procedures
Imaging for this study will include PET, MRI and a DEXA scan. The protocols for each are described
below.
Positron Emission Tomography (PET)
Dual Tracer PET scans (dtPET): A separate consent form for dual tracer PET administration and data
collection will be administered to all participants (IRB# 00033365 ). 11C-AcAc will be synthesized as
described previously.54, 55 PET scans will be performed on a GE Discovery PET/CT scanner with an
isotropic voxel size of 2 mm3, field-of-view of 25 cm, and axial field of 18 cm. Imaging with 11C-AcAc will
be conducted first with acquisition frames of 12x10 sec, 8x30 sec, and 1x4 min (total scan 10 min),
followed by a 50-min wash-out. FDG imaging is then conducted, using the time frames 12x10 sec, 8x30
sec, 6x4 min, and 3x10 min (total scan 60 min). Indwelling catheters will be placed in forearm veins for
the injection of 5-10 mci of tracer. Blood samples will be obtained at 3, 6, and 8 min after the 11C-AcAc
infusion and at 3, 8, 16, 24, 35 and 55 min after FDG infusion. Radioactivity in plasma samples is
counted in a gamma counter cross-calibrated with the PET scanner. PET images are preprocessed and
co-registered to each participant’s MR using a cross-modality 3D image fusion tool in PMOD 3.5. Co-
registered PET images are corrected for PVE with the modified Müller-Gartner method.56 Using the
PMOD 3.5 pixel-wise kinetic modeling tool, parametric images of CMRg and CMRa will be produced for
each participant. CMR quantification requires an arterial input function, determined by tracing regions of
interest (ROIs) on the internal carotid arteries with the aid of co-registered MR images as previously
validated.57 The calculated activity within the ROI will be corrected using the radioactivity of the plasma
samples obtained during image acquisition. The lumped constant will be set to 0.89 for the CMRg
calculation,58 and to 1.0 for the CMRa calculation. CMRg and CMRa will be expressed as mmol/100
g/min using the graphical Patlak model.59 CMRa will be corrected for loss of 5.9% of the dose of 11C-
AcAc that is catabolized to 11CO2.60 Key outcomes for analysis will be global 11C-acetoacetate uptake
and the PALZ PMOD index for 18F-FDG PET. 61
β-Amyloid PET Imaging (amyPET): All study participants will receive an amyloid PET scan. A
separate consent form for amyloid PET administration and data collection will be administered to all
participants (IRB# 00027675). Amyloid PET data will be used to confirm Alzheimer’s pathology and will
be added to the PET imaging repository of the ADCC. Participants will be injected with an iv bolus of up
to 15mCi (370 MBq) (+/- 10%) of 11C-Pittsburgh compound B (PiB, over 5-10 seconds), followed by a
40 min uptake period (+/- 10%). Brain emission images will be acquired continuously for a 30 min to
quantify amyloid uptake in MRI-defined regions, acquired in six 5-minute frames. Participants will be
contacted 24-72 hours after injection with 11C-PiB to inquire if any adverse events occurred within 24
hours of injection.
Study Protocol – BEATAD Draft 16-February-2018 Page 18Aβ deposition in the brain will be quantified by 11C-PiB uptake using standardized uptake volume ratio (SUVR) of 5 primary cortical areas (anterior cingulate; posterior cingulate/precuneus; frontal, lateral temporal and parietal cortex) relative to cerebellar gray matter. The unweighted average of these 5 regions will be used to determine amyloid positivity. Averages
TI = 3000 ms, FA = 90°, FOV = 240 mm, with a total of 36, 4 mm axial slices with a voxel size of
3.0x3.0x4.0mm. Slices will be collected in an ascending series and oriented in the transverse
plane. Quantitative perfusion maps (mL/100 gm tissue/min) for each voxel are computed as
previously described.64
Lumbar Puncture (LP) Procedure and CSF Assays: All participants will undergo LP before 11 a.m.
after fasting for ≥12 hours. Using a 22-gauge Sprotte needle to drip, up to 25ml of CSF will be
withdrawn into sterile polypropylene tubes. CSF will be transferred in 0.2 ml aliquots into pre-chilled
polypropylene tubes, frozen immediately on dry ice, and stored at -80°C until assay. CSF will be
analyzed for AD biomarkers (A42, tau, p-tau181) using the AlzBio3 multiplex assay (FujiRebio Europe).
Participants will be fed following the LP. The participant will rest in the recumbent position for 30
minutes post-LP. A recent Cochrane Review did not find that bed rest or IV fluid administration reduced
the risk of post-LP headache. Study participants or authorized caregivers will be contacted by phone
within 24 hours of the LP to inquire about any adverse events. Participants will sign a repository-
consent for storage of samples at the time of study consent.
Redox Proteomic/Lipidomic Assays: For the determination of free fatty acids (FFAs), 200 µl of CSF
will be extracted using chloroform/methanol according to a modified method of Bligh and Dyer,65 in
which the aqueous phase is adjusted to pH 3 or lower with HCl prior to extraction. Pentadecanoic and
heneicosanoic acids (Nu Chek Prep) will be added to each sample prior to extraction as internal
standards. The lipid extracts will be evaporated under a stream of argon, and the FFAs isolated by the
solid phase extraction method of Kaluzny et al.66 using 500 mg amino-SPE columns (Thermo
Scientific). FFAs will then be derivatized to their fatty acid methyl esters by treating with 1 ml of 14%
BF3 in methanol (Sigma-Aldrich) under Argon, and heating to 100°C for 10 minutes. After cooling, the
samples will be treated with 1 ml of hexane and 4 ml of saturated, aqueous NaCl. The hexane phase
will be transferred to a sample vial, and 1 μl analyzed on a TSQ Quantum XLS GC/MS/MS interfaced to
a Trace GC Ultra (Thermo Electron Corp.), automated by a TriPlus auto-sampler. Separation will be
accomplished using a 30 m × 0.25 mm diameter DB-WAX ETR column (Agilent Technologies) with a
0.25-μm coating as reported previously.67 Sphingolipids and ceramides will be analyzed using
published methods.68
DEXA Procedure: Participants will receive body fat assessments as part of the Pre-Diet and Post-Diet
Study Visits. Total fat mass, fat-free mass, and their relative distributions will be determined using dual
energy x-ray absorptiometry (DEXA) measurements. For this test, participants cannot have removable
metal objects on their body, such as snaps, belts, underwire bras, and jewelry. This test will last about
20-30 minutes.
Blood, CSF, and Tissue Biomarker Methods
Blood Collection Procedures/Measures: Participants will fast overnight prior to blood draw before 10
a.m. Whole blood will be collected and processed as described.69 Fasting blood for neuroendocrine
measures will be collected multiple times during the study period (visits 1, 3, 6-13). The total volume of
blood to be collected for neuroendocrine measures of stored blood ranges from 40-70mL (about 3-5
tablespoons) at cognitive Pre/Post Diet and Follow-up visits, and 25mL (about 2 tablespoons) at the
lumbar puncture visits. Included in this amount is blood to be stored for use in future studies.
Participants will sign a repository-consent for storage of samples at time of study consent. Additionally,
8.5mL of blood will be collected during the first week and again at 12 weeks for safety labs and
measurement of metabolic outcomes. The total amount of blood to be collected in this study will not
exceed 400mL over the 24-week study.
Study Protocol – BEATAD Draft 16-February-2018 Page 20Samples will be immediately placed on ice and spun within 30 minutes at 2200 rpm in a cold centrifuge for 15 minutes. Plasma, serum, and red blood cells will be aliquoted into separate storage tubes and flash frozen at -80°C until assays are analyzed. The following assays will be performed. Metabolic assays: Metabolic measures will be assessed at the Pre-Diet, Mid-Diet and Post-Diet visits. These will include, glucose, insulin, lipids (HDL, LDL, VLDL, and triglycerides) will be measured by LabCorp diagnostics. APOE genotyping: Additional blood (up to 10 ml, Visit 2) will be drawn for confirmation and validation of ApoE genotyping by the Laboratory of Donald Bowden, PhD at Wake Forest School of Medicine. Participants that have already had APOE genotyping done as a part of another ADCC stuidy will not have to repeat this procedure. The total amount of blood to be drawn at this visit, including blood for the repository, mitochondrial assay and genotyping, is 70 ml (~5 tablespoons). Mitochondrial respiration assays: To determine bioenergetic profiles of intact cells, we will assess basal respiration (Basal-OCR), maximal respiration (MAX), ATP coupled respiration (ATP-OCR), proton leak (Leak), and non- mitochondrial respiration (non-mito).70-72 The spare respiratory capacity (SRC) will be calculated by subtracting Basal-OCR from MAX. A Seahorse XF96e will be used to run samples in triplicate. Basal- OCR measurements will be performed with standard fuel conditions designed to examine glucose oxidation (pyruvate and glucose). Oligomycin will be added to block ATP synthase. The resultant decrease in oxygen consumption (ATP-OCR) reflects the amount of oxygen consumed to fuel the conversion of ADP to ATP. FCCP will be added to uncouple mitochondria and induce maximal oxygen consumption. The resultant increase in oxygen consumption represents MAX. Antimycin-A and rotenone will be added to block electron transport chain activity. Residual respiration is non- mitochondrial. Oxygen consumption not linked to ATP synthesis (Leak) will be measured by subtracting non-mito OCR from the rate left after the blockage of ATP synthase by oligomycin. Epigenomics/Transcriptomics Read Mapping/Counting: Illumina sequencing runs will be processed to generate FastQ files using the Illumina provided configureBclToFastq.pl script to automate running CASAVA, using default parameters for removal of sequencing reads failing the chastity filter and mismatches in the barcode read. Reads will be trimmed for QC using Btrim (5 base sliding window average with Q > 15)73 and to remove any adaptor sequence using ea-utils. The current Ensembl Homo Sapiens reference file, annotations and Bowtie2 indexes will be used for mapping of the sequencing reads to the genome and read counting. Bowtie2 and TopHat2 will be used to map the sequencing reads to the genome using a mate-inner- distance of 100 bp and ‘firststrand’ options.74,75 Following alignment, read counts per gene will be obtained using HTSeq(88).Pre-Processing and QC of mRNA Raw Count Data will be performed in R using Bioconductor packages. Counts will be converted to Counts Per Million (CPM) using the cpm function of the edgeR package,76 and all features with CPM ≤ 0.25 in ≥90% will be removed. We will use the biomaRt package and the Ensembl BioMart database to obtain Entrez Gene IDs, Gene Symbols, genome coordinates, gene length and percent GC content for detectable features. We will perform differential isoform expression analysis.77 To use linear modeling in R, we will transform the raw count data to log CPM,78 and use the Trimmed Mean of M-values (TMM) normalization method79 implemented in the calcNormFactors function in the edgeR Bioconductor package.80 We will adjust for flow cell effects. Pre-Processing and QC of DNA Methylation Data: The raw DNA methylation microarray data will be pre-processed with published pipelines for expression and methylation implemented in R using Bioconductor packages. The final methylation value for each probe is computed as the M-value (log ratio of methylated to unmethylated intensity).81 Filters applied to the probes for the CpG sites include detected methylation levels in
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